Publications by authors named "Bernd Reif"

121 Publications

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability.

J Biol Chem 2020 12 22;295(52):18474-18484. Epub 2020 Oct 22.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und UmweltInstitute of Structural Biology (STB), Neuherberg, Germany; Munich Center for Integrated Protein Science (CIPS-M) at the Dept. of Chemistry, Technische Universität München (TUM), Garching, Germany. Electronic address:

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.
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http://dx.doi.org/10.1074/jbc.RA120.016006DOI Listing
December 2020

Domain Interactions Determine the Amyloidogenicity of Antibody Light Chain Mutants.

J Mol Biol 2020 11 13;432(23):6187-6199. Epub 2020 Oct 13.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85748 Garching, Germany. Electronic address:

In antibody light chain amyloidosis (AL), mutant light chains (LCs) or their variable domains (Vs) form fibrils, which accumulate in organs and lead to their failure. The molecular mechanism of this disease is still poorly understood. One of the key open issues is whether the mutant Vs and LCs differ in fibril formation. We addressed this question studying the effects of the V mutations S20N and R61A within the isolated V domain and in the full-length LC scaffold. Both V variants readily form fibrils. Here, we find that in the LC context, the S20N variant is protected from fibril formation while for LC R61A fibril formation is even accelerated compared to V R61A. Our analyses revealed that the partially unfolded state of the V R61A domain destabilizes the C domain by non-native interactions, in turn leading to a further unfolding of the V domain. In contrast, the folded mutant V S20N and V wt form native interactions with C. These are beneficial for LC stability and promote amyloid resistance. Thus the effects of specific mutations on the V fold can have opposing effects on LC domain interactions, stability and amyloidogenicity.
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http://dx.doi.org/10.1016/j.jmb.2020.10.005DOI Listing
November 2020

Small molecule induced toxic human-IAPP species characterized by NMR.

Chem Commun (Camb) 2020 Nov 2;56(86):13129-13132. Epub 2020 Oct 2.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.
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http://dx.doi.org/10.1039/d0cc04803hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641245PMC
November 2020

Solid state NMR assignments of a human λ-III immunoglobulin light chain amyloid fibril.

Biomol NMR Assign 2021 Apr 18;15(1):9-16. Epub 2020 Sep 18.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit Und Umwelt, Institute of Structural Biology, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

The aggregation of antibody light chains is linked to systemic light chain (AL) amyloidosis, a disease where amyloid deposits frequently affect the heart and the kidney. We here investigate fibrils from the λ-III FOR005 light chain (LC), which is derived from an AL-patient with severe cardiac involvement. In FOR005, five residues are mutated with respect to its closest germline gene segment IGLV3-19 and IGLJ3. All mutations are located close to the complementarity determining regions (CDRs). The sequence segments responsible for the fibril formation are not yet known. We use fibrils extracted from the heart of this particular amyloidosis patient as seeds to prepare fibrils for solid-state NMR. We show that the seeds induce the formation of a specific fibril structure from the biochemically produced protein. We have assigned the fibril core region of the FOR005-derived fibrils and characterized the secondary structure propensity of the observed amino acids. As the primary structure of the aggregated patient protein is different for every AL patient, it is important to study, analyze and report a greater number of light chain sequences associated with AL amyloidosis.
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http://dx.doi.org/10.1007/s12104-020-09975-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973639PMC
April 2021

Mapping the Binding Interface of PET Tracer Molecules and Alzheimer Disease Aβ Fibrils by Using MAS Solid-State NMR Spectroscopy.

Chembiochem 2020 09 19;21(17):2495-2502. Epub 2020 May 19.

Munich Center for Integrated Protein Science (CIPS-M) Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747, Garching, Germany.

Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid-state NMR spectroscopy to characterize Aβ-PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1-40) fibrillar aggregates. The same Aβ(1-40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules C-methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical C-detected as well as proton-detected spectra of protonated and perdeuterated samples with back-substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical-shift perturbations were observed in the loop region involving residues Gly25-Lys28 and Ile32-Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β-sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure.
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http://dx.doi.org/10.1002/cbic.202000143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496087PMC
September 2020

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action.

Angew Chem Int Ed Engl 2020 03 28;59(14):5771-5781. Epub 2020 Jan 28.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Institute of Structural Biology, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a β-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.
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http://dx.doi.org/10.1002/anie.201914559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154662PMC
March 2020

The structure and oxidation of the eye lens chaperone αA-crystallin.

Nat Struct Mol Biol 2019 12 2;26(12):1141-1150. Epub 2019 Dec 2.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.
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http://dx.doi.org/10.1038/s41594-019-0332-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115824PMC
December 2019

Accessing Methyl Groups in Proteins via H-detected MAS Solid-state NMR Spectroscopy Employing Random Protonation.

Sci Rep 2019 11 4;9(1):15903. Epub 2019 Nov 4.

Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747, Garching, Germany.

We recently introduced RAP (reduced adjoining protonation) labelling as an easy to implement and cost-effective strategy to yield selectively methyl protonated protein samples. We show here that even though the amount of HO employed in the bacterial growth medium is rather low, the intensities obtained in MAS solid-state NMR H,C correlation spectra are comparable to spectra obtained for samples in which α-ketoisovalerate was employed as precursor. In addition to correlations for Leu and Val residues, RAP labelled samples yield also resonances for all methyl containing side chains. The labelling scheme has been employed to quantify order parameters, together with the respective asymmetry parameters. We obtain a very good correlation between the order parameters measured using a GlcRAP (glucose carbon source) and a α-ketoisovalerate labelled sample. The labelling scheme holds the potential to be very useful for the collection of long-range distance restraints among side chain atoms. Experiments are demonstrated using RAP and α-ketoisovalerate labelled samples of the α-spectrin SH3 domain, and are applied to fibrils formed from the Alzheimer's disease Aβ peptide.
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http://dx.doi.org/10.1038/s41598-019-52383-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828780PMC
November 2019

Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16.

Redox Biol 2020 01 3;28:101316. Epub 2019 Sep 3.

Center for Molecular Medicine, Molecular Cancer Research, University Medical Center Utrecht, Universiteitsweg 100, 3584CG, Utrecht, The Netherlands. Electronic address:

The tumor suppressor p16 induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16 is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16 in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16 is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16 undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16 amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16 through the formation of amyloid fibrils.
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http://dx.doi.org/10.1016/j.redox.2019.101316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812003PMC
January 2020

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR.

J Biomol NMR 2019 Nov 12;73(10-11):625-631. Epub 2019 Sep 12.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH, CHD, CHD) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.
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http://dx.doi.org/10.1007/s10858-019-00274-0DOI Listing
November 2019

Determination of methyl order parameters using solid state NMR under off magic angle spinning.

J Biomol NMR 2019 Sep 12;73(8-9):471-475. Epub 2019 Aug 12.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

Quantification of dipolar couplings in biological solids is important for the understanding of dynamic processes. Under Magic Angle Spinning (MAS), order parameters are normally obtained by recoupling of anisotropic interactions involving the application of radio frequency pulses. We have recently shown that amide backbone order parameters can be estimated accurately in a spin-echo experiment in case the rotor spinning angle is slightly mis-calibrated. In this work, we apply this method to determine methyl order parameters in a deuterated sample of the SH3 domain of chicken α-spectrin in which the methyl containing side chains valine and leucine are selectively protonated.
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http://dx.doi.org/10.1007/s10858-019-00253-5DOI Listing
September 2019

Probing transient non-native states in amyloid beta fiber elongation by NMR.

Chem Commun (Camb) 2019 Apr;55(31):4483-4486

Biophysics, University of Michigan, Ann Arbor, MI 48109-1055, USA.

Using NMR to probe transient binding of Aβ1-40 monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19-K28 and a lesser degree of interaction near the C-terminus (L34-G37). This represents a shift away from the KLVFFA recognition sequence (residues 16-21) currently used for inhibitor design.
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http://dx.doi.org/10.1039/c9cc01067jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544147PMC
April 2019

Accurate Determination of H- N Dipolar Couplings Using Inaccurate Settings of the Magic Angle in Solid-State NMR Spectroscopy.

Angew Chem Int Ed Engl 2019 03 27;58(13):4286-4290. Epub 2019 Feb 27.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

Magic-angle spinning (MAS) is an essential ingredient in a wide variety of solid-state NMR experiments. The standard procedures to adjust the rotor angle are not highly accurate, resulting in a slight misadjustment of the rotor from the magic angle ( ) on the order of a few millidegrees. This small missetting has no significant impact on the overall spectral resolution, but is sufficient to reintroduce anisotropic interactions. Shown here is that site-specific H- N dipolar couplings can be accurately measured in a heavily deuterated protein. This method can be applied at arbitrarily high MAS frequencies, since neither rotor synchronization nor particularly high radiofrequency field strengths are required. The off-MAS method allows the quantification of order parameters for very dynamic residues, which often escape an analysis using existing methods.
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http://dx.doi.org/10.1002/anie.201814314DOI Listing
March 2019

The Antibody Light-Chain Linker Regulates Domain Orientation and Amyloidogenicity.

J Mol Biol 2018 12 8;430(24):4925-4940. Epub 2018 Nov 8.

Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Lichtenbergstr, 4, 85748 Garching, Germany. Electronic address:

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the V and C domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of V and C to each other are of major importance for maintaining the structural integrity of the full-length LC.
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http://dx.doi.org/10.1016/j.jmb.2018.10.024DOI Listing
December 2018

A single residue switch reveals principles of antibody domain integrity.

J Biol Chem 2018 11 18;293(44):17107-17118. Epub 2018 Sep 18.

From the Center for Integrated Protein Science Munich, Departments of Chemie and

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the C2 domain. We found that compared with other Ig domains, the isolated C2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a C2 variant substantially increases the melting temperature by ∼14 °C relative to C2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of C2. The results revealed that Lys is key for the formation of three secondary structure elements: the very C-terminal β-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys and the nearby α-helix, is important for stabilizing the C2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.
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http://dx.doi.org/10.1074/jbc.RA118.005475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222096PMC
November 2018

Overcoming Volume Selectivity of Dipolar Recoupling in Biological Solid-State NMR Spectroscopy.

Angew Chem Int Ed Engl 2018 10 31;57(44):14514-14518. Epub 2018 Jul 31.

Munich Center for Integrated Protein Science (CIPS-M) at, Department Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, 85747, Garching, Germany.

Dipolar recoupling in solid-state NMR is an essential method for establishing correlations between nuclei that are close in space. In applications on protein samples, the traditional experiments like ramped and adiabatic DCP suffer from the fact that dipolar recoupling occurs only within a limited volume of the sample. This selection is dictated by the radiofrequency (rf) field inhomogeneity profile of the excitation solenoidal coil. We employ optimal control strategies to design dipolar recoupling sequences with substantially larger responsive volume and increased sensitivity. We show that it is essential to compensate for additional temporal modulations induced by sample rotation in a spatially inhomogeneous rf field. Such modulations interfere with the pulse sequence and decrease its performance. Using large-scale optimizations we developed pulse schemes for magnetization transfer from amide nitrogen to carbonyl (NCO) as well as aliphatic carbons (NCA). Our experiments yield a signal intensity increased by a factor of 1.5 and 2.0 for NCA and NCO transfers, respectively, compared to conventional ramped DCP sequences. Consistent results were obtained using several biological samples and NMR instruments.
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http://dx.doi.org/10.1002/anie.201805002DOI Listing
October 2018

The neuronal S100B protein is a calcium-tuned suppressor of amyloid-β aggregation.

Sci Adv 2018 06 29;4(6):eaaq1702. Epub 2018 Jun 29.

Biosystems and Integrative Sciences Institute, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.

Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer's disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface-catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.
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http://dx.doi.org/10.1126/sciadv.aaq1702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025902PMC
June 2018

hIAPP forms toxic oligomers in plasma.

Chem Commun (Camb) 2018 May;54(43):5426-5429

Institute for Advanced Study, Technische Universität München, 85748 Garching, Germany.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.
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http://dx.doi.org/10.1039/c8cc03097aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970100PMC
May 2018

Reconstitution of Isotopically Labeled Ribosomal Protein L29 in the 50S Large Ribosomal Subunit for Solution-State and Solid-State NMR.

Methods Mol Biol 2018 ;1764:87-100

Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Garching, Germany.

Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S ribosomal subunit. In particular, we focus on the 63-residue ribosomal protein L29 (~7 kDa), which is located at the exit of the tunnel of the large 50S ribosomal subunit (~1.5 MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP).
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http://dx.doi.org/10.1007/978-1-4939-7759-8_6DOI Listing
February 2019

Physical basis of amyloid fibril polymorphism.

Nat Commun 2018 02 16;9(1):699. Epub 2018 Feb 16.

Institute of Protein Biochemistry, Ulm University, 89081, Ulm, Germany.

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide-peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures.
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http://dx.doi.org/10.1038/s41467-018-03164-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816019PMC
February 2018

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

Sci Rep 2018 01 18;8(1):1116. Epub 2018 Jan 18.

Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.
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http://dx.doi.org/10.1038/s41598-017-18807-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773570PMC
January 2018

Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate.

Elife 2017 11 17;6. Epub 2017 Nov 17.

Institute for Advanced Study, Technische Universität München, Garching, Germany.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.
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http://dx.doi.org/10.7554/eLife.31226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706959PMC
November 2017

Radiofrequency fields in MAS solid state NMR probes.

J Magn Reson 2017 11 6;284:20-32. Epub 2017 Sep 6.

Munich Center for Integrated Protein Science (CIPS-M) at Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85747 Garching, Germany; Department of Chemistry, Technical University of Munich, Lichtenbergstr. 4, 85747 Garching, Germany; Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.

We present a detailed analysis of the radiofrequency (RF) field over full volume of a rotor that is generated in a solenoid coil. On top of the usually considered static distribution of amplitudes along the coil axis we describe dynamic radial RF inhomogeneities induced by sample rotation. During magic angle spinning (MAS), the mechanical rotation of the sample about the magic angle, a spin packet travels through areas of different RF fields and experiences periodical modulations of both the RF amplitude and the phase. These modulations become particularly severe at the end regions of the coil where the relative RF amplitude varies up to ±25% and the RF phase changes within ±30°. Using extensive numerical simulations we demonstrate effects of RF inhomogeneity on pulse calibration and for the ramped CP experiment performed at a wide range of MAS rates. In addition, we review various methods to map RF fields using a B gradient along the sample (rotor axis) for imaging purposes. Under such a gradient, a nutation experiment provides directly the RF amplitude distribution, a cross polarization experiment images the correlation of the RF fields on the two channels according to the Hartmann-Hahn matching condition, while a spin-lock experiment allows to calibrate the RF amplitude employing the rotary resonance recoupling condition. Knowledge of the RF field distribution in a coil provides key to understand its effects on performance of a pulse sequence at the spectrometer and enables to set robustness requirements in the experimental design.
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http://dx.doi.org/10.1016/j.jmr.2017.09.002DOI Listing
November 2017

Comparative Study of REDOR and CPPI Derived Order Parameters by H-Detected MAS NMR and MD Simulations.

J Phys Chem B 2017 09 12;121(37):8719-8730. Epub 2017 Sep 12.

Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM) , Lichtenbergstr. 4, 85747 Garching, Germany.

The measurement of dipolar couplings among directly bonded nuclei yields direct information on the amplitude of dynamic processes in the solid-state. For a reliable motional analysis using, e.g., the model-free approach, a correct quantification of the absolute values of these order parameters is absolutely essential. In the absence of a reference value for the rigid limit, too low dipolar coupling values might be misinterpreted as motion. Therefore, a detailed understanding of the effects that influence the quantification of the experimental order parameters is necessary. We compare here REDOR and CPPI derived order parameters assessed in H-detected experiments, and discuss the influence of remote protons and rf inhomogeneity on the extracted dipolar coupling constant for MAS rotation frequencies in the range 20-100 kHz. Experimental results are furthermore compared with the order parameter obtained from a molecular dynamics simulation. We find that fast magic-angle spinning up to 100 kHz can yield artifact-free REDOR based H,N order parameters for perdeuterated and 100% amide back-exchanged proteins, and potentially even in uniformly protonated samples. We believe that awareness of systematic errors introduced by the measurement and in the analysis of order parameters will yield a better understanding of the dynamic properties of a protein derived from solid-state NMR observables.
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http://dx.doi.org/10.1021/acs.jpcb.7b06812DOI Listing
September 2017

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins.

Sci Rep 2017 08 7;7(1):7444. Epub 2017 Aug 7.

Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.

MAS solid-state NMR is capable of determining structures of protonated solid proteins using proton-detected experiments. These experiments are performed at MAS rotation frequency of around 110 kHz, employing 0.5 mg of material. Here, we compare H, C correlation spectra obtained from protonated and deuterated microcrystalline proteins at MAS rotation frequency of 111 kHz, and show that the spectral quality obtained from deuterated samples is superior to those acquired using protonated samples in terms of resolution and sensitivity. In comparison to protonated samples, spectra obtained from deuterated samples yield a gain in resolution on the order of 3 and 2 in the proton and carbon dimensions, respectively. Additionally, the spectrum from the deuterated sample yields approximately 2-3 times more sensitivity compared to the spectrum of a protonated sample. This gain could be further increased by a factor of 2 by making use of stereospecific precursors for biosynthesis. Although the overall resolution and sensitivity of H, C correlation spectra obtained using protonated solid samples with rotation frequencies on the order of 110 kHz is high, the spectral quality is still poor when compared to the deuterated samples. We believe that experiments involving large protein complexes in which sensitivity is limiting will benefit from the application of deuteration schemes.
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http://dx.doi.org/10.1038/s41598-017-07253-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547042PMC
August 2017

MAK33 antibody light chain amyloid fibrils are similar to oligomeric precursors.

PLoS One 2017 26;12(7):e0181799. Epub 2017 Jul 26.

Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische Universität München (TUM), Germany.

Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0181799PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528828PMC
October 2017

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Sci Rep 2017 03 13;7:44041. Epub 2017 Mar 13.

Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg 85764, Germany.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
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http://dx.doi.org/10.1038/srep44041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347123PMC
March 2017

Epigallocatechin-3-gallate preferentially induces aggregation of amyloidogenic immunoglobulin light chains.

Sci Rep 2017 01 27;7:41515. Epub 2017 Jan 27.

Center for Integrated Protein Science at Department Chemie, Technische Universität München Lichtenbergstrasse 4, 85747 Garching, Germany.

Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs.
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http://dx.doi.org/10.1038/srep41515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5269747PMC
January 2017

Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity.

Solid State Nucl Magn Reson 2016 Jun-Jul;76-77:7-14. Epub 2016 Mar 15.

Munich Center for Integrated Protein Science (CIPSM) at Department of Chemie, Technische Universität München (TUM), Lichtenbergstr. 4, D-85747 Garching, Germany; Helmholtz-Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (HMGU), Ingolstädter Landstr. 1, D-85764 Neuherberg, Germany. Electronic address:

In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained.
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http://dx.doi.org/10.1016/j.ssnmr.2016.03.005DOI Listing
December 2017